Method for inhibiting microbial growth in liquid nutrients

ABSTRACT

A method for inhibiting growth of microbes in a liquid having a pH equal to or greater than about 2.5 by using a filter bed assembly or filter for filtering the liquid having a metal-ion sequestering agent for removing designated metal ion from the liquid. The liquid is passed through the filter bed or filter for a sufficient time so as to substantially reduce the designated metal ion from the liquid.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Continuation-in-Part of U.S. patent application Ser. No.10/936,929 filed Sep. 9, 2004 entitled CONTAINER FOR INHIBITINGMICROBIAL GROWTH IN LIQUID NUTRIENTS by David L. Patton et al., which isa Continuation-in-Part of application Ser. No. 10/823,446 filed Apr. 13,2004 now U.S. Pat. No. 7,258,786 entitled CONTAINER FOR INHIBITINGMICROBIAL GROWTH IN LIQUID NUTRIENTS by David L. Patton, et al.

Reference is also made to commonly assigned pending U.S. patentapplication Ser. No. 10/985,378 filed Nov. 10, 2004 entitled CONTAINERFOR INHIBITING MICROBIAL GROWTH IN LIQUID NUTRIENTS by David L. Patton,Joseph F. Bringley, Richard W. Wien, John M. Pochan, Yannick J. F. Leratand Narashimharao Dontula and pending U.S. patent application Ser. No.10/985,377 filed Nov. 10, 2004 entitled CONTAINER FOR INHIBITINGMICROBIAL GROWTH IN LIQUID NUTRIENTS by David L. Patton, Joseph F.Bringley, Richard W. Wien, John M. Pochan, Yannick J. F. Lerat andWilliam J. Harrison the disclosures of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a fluid container having a metal-ionsequestering agent for removing bio-essential metal ions from a liquidnutrient for inhibiting growth of microbes in the liquid nutrient.

BACKGROUND OF THE INVENTION

It has been recognized that small concentrations of metal ions play animportant role in biological processes. For example, Mn, Mg, Fe, Ca, Zn,Cu and Al are essential bio-metals, and are required for most, if notall, living systems. Metal ions play a crucial role in oxygen transportin living systems, and regulate the function of genes and replication inmany cellular systems. Calcium is an important structural element in thelife of bacteria regulating enzyme activity. Mn, Mg, Cu and Fe areinvolved in metabolism and enzymatic processes. At high concentrations,metals may become toxic to living systems and the organism mayexperience disease or illness if the level cannot be controlled. As aresult the availability and concentrations, of metal ions in biologicalenvironments is a major factor in determining the abundance, growth-rateand health of plant, animal and micro-organism populations.

It has also been recognized that iron is an essential biologicalelement, and that all living organisms require iron for survival andreplication. Although, the occurrence and concentration of iron isrelatively high on the earth's surface, the availability of “free” ironis severely limited by the extreme insolubility of iron in aqueousenvironments. As a result, many organisms have developed complex methodsof procuring “free” iron for survival and replication.

Articles, such as food and beverage containers are needed that are ableto improve food quality, to increase shelf-life, to protect frommicrobial contamination, and to do so in a manner that is safe for theuser of such items and that is environmentally clean while providing forthe general safety and health of the public. Materials and methods areneeded to prepare articles having antimicrobial properties that areless, or not, susceptible to microbial resistance. Methods are neededthat are able to target and remove specific, biologically important,metal ions while leaving intact the concentrations of beneficial metalions.

During the process of filling containers with certain beverages andfoods, air borne pathogens enter the containers after the flashpasteurization or pasteurization part of the process. These pathogenssuch as yeast, spores, bacteria, etc. will grow in the nutrient richbeverage or food, ruining the taste or even causing hazardousmicrobiological contamination. While some beverages are packaged byaseptic means or by utilizing preservatives, many other beverages, forexample fruit juices, teas and isotonic drinks are “hot-filled”.“Hot-filling” involves the filling of a container with a liquid beveragehaving some elevated temperature (typically at about 180-200° F.). Thecontainer is capped and allowed to cool, producing a partial vacuumtherein. The process of hot filling of beverages and foods is used tokill the pathogens which enter the container during the filling of thebeverage or food containers. Hot filling requires containers be made ofcertain materials or constructed in a certain fashion such as thickerwalls to withstand the hot filling process. The energy required for hotfilling adds to the cost of the filling process. Temperatures requiredfor hot filling have a detrimental effect on the flavor of the beverage.Other methods of filling such as aseptic filling require large capitalexpenditures and maintaining class 5 clean room conditions.

U.S. Pat. No. 5,854,303 discloses a polymeric material incorporating apolyvalent cation chelating agent in an amount effective to inhibit thegrowth of a protozoan on the surface of contact lenses and in other eyecare products.

PROBLEM TO BE SOLVED BY THE INVENTION

The present invention is directed to the problem of the growth ofmicro-organism in liquids that adversely affects food quality,shelf-life, and to protect the liquid from microbial contamination byproviding storage/holding tanks or passed the liquid through a filterdevice.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a method for inhibiting growth of microbes in a liquid having apH equal to or greater than about 2.5, comprising the steps of:

a. providing a filter bed assembly for filtering the liquid, the filterbed assembly having a metal-ion sequestering agent for removingdesignated metal ion from the liquid, the filter assembly having aninlet and an outlet for allowing the liquid to enter and leave thefilter assembly; and

b. causing the liquid to enter the fluid bed assembly through an inletpass through the filter bed and out an outlet for a sufficient time soas to substantially reduce the designated metal ion from the liquid.

In accordance with another aspect of the present invention there isprovided a method for inhibiting growth of microbes in a liquid having apH equal to or greater than about 2.5, comprising the steps of:

a. providing a filter assembly for filtering the liquid, the filterassembly having a filter having a metal-ion sequestering agent forremoving designated metal ion from the liquid, the filter assemblyhaving an inlet and an outlet for allowing the liquid to enter and leavethe filter assembly; and

b. causing the liquid to enter the filter assembly through the inletpass through the filter and out the outlet for a sufficient time so asto substantially reduce the designated metal ion from the liquid.

These and other aspects, objects, features and advantages of the presentinvention will be more clearly understood and appreciated from a reviewof the following detailed description of the preferred embodiments andappended claims and by reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the detailed description of the preferred embodiments of theinvention presented below, reference is made to the accompanyingdrawings in which:

FIG. 1 illustrates a cross section of a fluid container made inaccordance with the prior art;

FIG. 2 is an enlarged partial cross sectional view of a portion of thecontainer of FIG. 1 illustrating a “free” iron ion sequestering agent;

FIG. 3 is a view similar to FIG. 2 illustrating a container made inaccordance with the present invention;

FIG. 4 illustrates a bottle with a bottle cap also made in accordancewith the present invention;

FIG. 5 is a schematic top plan view of the bottle and cap of FIG. 4;

FIG. 6 is an enlarged partial cross sectional view of the bottle and captaken along line 6-6 of FIG. 5;

FIG. 7 is a schematic view of a projecting member extending from amodified cap of FIG. 5 also made in accordance with the presentinvention;

FIG. 8 is an enlarged cross sectional view of the projecting member ofFIG. 7 as taken along line 8-8;

FIG. 9 is a schematic view of another embodiment of the presentinvention illustrating one method for applying a coating to the interiorsurface of a bottle made in accordance with the present invention;

FIG. 10 is an enlarged partial cross sectional view of a portion of thebottle of FIG. 9 illustrating the sprayed coating of the ionsequestering agent;

FIG. 11 is a schematic view of another fluid container made inaccordance with the present invention such as a juice box;

FIG. 12 is an enlarged partial cross sectional view of the juice boxtaken along line 12-12 of FIG. 11;

FIG. 13 is a schematic view of yet another fluid container such as astand up pouch made in accordance with the present invention;

FIG. 14 is an enlarged partial cross sectional view of the stand uppouch taken along line 14-14 of FIG. 13;

FIG. 15 is a schematic view of still another embodiment of a fluidcontainer such as a bag also made in accordance with the presentinvention;

FIG. 16 is an enlarged partial cross sectional view of a portion of thebag of FIG. 15 as indicated by circle 16;

FIG. 17 is a cross-sectional view of a web that can be used in themanufacture of a box, pouch or bag showing a coating assembly forcoating a hydrophilic layer containing a metal-ion sequestering agent;

FIG. 18 is a schematic view of yet another fluid container, such as acan, made in accordance with the present invention;

FIG. 19 is a cross sectional view of FIG. 18 as taken along line 19-19;

FIG. 20 is a cross sectional view of a filter assembly made inaccordance with the present invention;

FIG. 21 is a cross sectional view of a fluid bed ion exchange assemblymade in accordance with the present invention; and

FIG. 22 is an enlarged partial view of a portion of the fluid bed ionexchange assembly of FIG. 21 as identified by circle 22 illustrating ametal-ion sequestering agent.

FIG. 23 is a schematic top view of the web and device of FIG. 24.

FIG. 24 is a schematic view of electro-photographic device for applyinga pattern of metal ion chelating agents to a web, such as is used tomake a box, pouch or bag in accordance with the present invention.

FIG. 25 is a graph illustrating iron chelation of model beverages.

FIG. 26 is a graph illustrating rates of iron removal from 17.2% w/wOcean Spray™ Apple Juice Concentrate.

FIG. 27 is a bar graph illustrating fungostatic and fungicidal effects.

DETAILED DESCRIPTION OF THE INVENTION

The growth of microbes in an article such as a fluid containercontaining a liquid nutrient comprising a liquid nutrient can beinhibited by placing metal-ion sequestering agents, as described in U.S.patent application Ser. No. 10/822,940 filed Apr. 13, 2004 entitledDERIVATIZED NANOPARTICLES COMPRISING METAL-ION SEQUESTRANT by Joseph F.Bringley, and U.S. patent application Ser. No. 10/822,929 filed Apr. 13,2004 entitled COMPOSITION OF MATTER COMPRISING POLYMER AND DERIVATIZEDNANOPARTICLES by Joseph F. Bringley et al. capable of removing adesignated metal ion for example, Mn, Mg, Fe, Ca, Zn, Cu and Al fromsaid liquid nutrients, in contact with the nutrient. Intimate contact isachieved by incorporating the metal-ion sequestering agent as anintegral part of the support structure of the article. For example, onecan control the concentration of “free” iron in the liquid nutrient heldby the article by placing an iron sequestering agent in the walls of thecontainer, which in turn controls the growth rates, and abundance ofmicro-organisms. The articles of the invention further contain aneffective amount of an antimicrobial agent, which quickly reduces thepopulation of microbes to a manageable level, and insures theeffectiveness of metal-ion sequestering or binding agents. The invention“starves” the remaining micro-organisms of minute quantities ofessential nutrients (metal-ions) and hence limits their growth andreduces the risk due to bacterial, viral and other infectious diseases.The article, such as a container, may be used for holding a food orbeverage.

The term inhibition of microbial-growth, or a material which “inhibits”microbial growth, is used by the authors to mean materials, which eitherprevent microbial growth, or subsequently kills microbes so that thepopulation is within acceptable limits, or materials, whichsignificantly retard the growth processes of microbes or maintain thelevel or microbes to a prescribed level or range. The prescribed levelmay vary widely depending upon the microbe and its pathogenicity;generally it is preferred that harmful organisms are present at no morethan 10 organisms/ml and preferably less than 1 organism/ml.Antimicrobial agents which kill microbes or substantially reduce thepopulation of microbes are often referred to as biocidal materials,while materials which simply slow or retard normal biological growth arereferred to as biostatic materials. The preferred impact upon themicrobial population may vary widely depending upon the application, forpathogenic organisms (such as E. coli O157:H7) a biocidal effect is morepreferred, while for less harmful organisms a biostatic impact may bepreferred. Generally, it is preferred that microbiological organismsremain at a level which is not harmful to the consumer or user of thatparticular article

Metal-ion sequestering agents may be incorporated into articles byplacing the metal-ion sequestering agents on the surface of the article,or by putting the metal-ion sequestering agents within the materialsused to form the article. In all instances, the metal-ion sequesteringagents must be capable of contacting the food or beverage held by thecontainer.

Referring to FIG. 1, there is illustrated a cross-sectional view of atypical prior art container. In the embodiment illustrated, thecontainer comprises a bottle 5 holding a liquid nutrient 10, for examplean isotonic liquid. Drinks such as Gatorade™ or PowerAde™ are examplesof isotonic drinks/liquids. The container 5 may be made of one or morelayers of a plastic polymer using various molding processes known bythose skilled in the art. Examples of polymers used in the manufactureof bottles are PET (polyethylene terephthalate), PP (polypropylene),LDPE (low density polyethylene) and HDPE (high density polyethylene).FIG. 2 illustrates a plastic bottle 5 formed using two differentpolymeric layers 15 and 20. However it is to be understood that thecontainer 5 may comprise any desired number of layers.

A fluid container made in accordance with the present invention isespecially useful for containing a liquid nutrient having a pH equal toor greater than about 2.5. The container is designed to have an interiorsurface having a metal-ion sequestering agent for removing a designatedmetal ion from a liquid nutrient for inhibiting growth of microbes insaid liquid nutrient. It is preferred that the metal-ion sequestrant isimmobilized within the materials forming the container or is immobilizedwithin a polymeric layer directly in contact with the beverage or liquidnutrient. It is further preferred that the metal-ion sequestering agentis immobilized on the surface(s) of said container. This is importantbecause metal-ion sequestrants that are not immobilized may diffusethrough the material or polymeric layers of the container and dissolveinto the contents of the beverage. Metal ions complexed by dissolvedsequestrants will not be sequestered within the surfaces of thecontainer but may be available for use by micro-organisms.

It is preferred that the sequestering agent is immobilized on thesurface(s) of said container and has a high-affinity for biologicallyimportant metal ions such as Mn, Mg, Zn, Cu and Fe. It is furtherpreferred that the immobilized sequestering agent has a high-selectivityfor biologically important metal ions such as Mn, Mg, Zn, Cu and Fe. Itis preferred that said sequestering agent has a high-selectively forcertain metal ions but a low-affinity for at least one other ion. It isfurther preferred that said certain metal ions comprises Mn, Mg, Zn, Cuand Fe and said other at least one ion comprises calcium. This ispreferred because some metal ions such as calcium, sodium and potassiummay be beneficial to the taste and quality of the food, and are usuallyvery highly abundant in foodstuffs and in liquid extrudates offoodstuffs. It is preferred that said metal-ion sequestering agent isimmobilized on the surface(s) of said container and has a stabilityconstant greater than 10¹⁰ with iron (III), more preferably greater than10²⁰ with iron (III), and most preferably greater than 10³⁰ with iron(III). This is preferred because iron is an essential nutrient forvirtually all micro-organisms, and sequestration of iron may mostbeneficially limit the growth of micro-organisms.

In a particularly preferred embodiment, the invention provides a fluidcontainer wherein said metal-ion sequestering agent comprisesderivatized nanoparticles comprising inorganic nanoparticles having anattached metal-ion sequestrant, wherein said inorganic nanoparticleshave an average particle size of less than 200 nm and the derivatizednanoparticles have a stability constant greater than 10¹⁰ with iron(III). It is preferred that the inorganic nanoparticles have an averageparticle size of less than 100 nm. It is preferred that said metal-ionsequestrant is attached to the nanoparticle by reacting the nanoparticlewith a silicon alkoxide intermediate of the sequestrant having thegeneral formula:Si(OR)_(4-x)R′_(x);wherein x is an integer from 1 to 3;

-   R is an alkyl group; and-   R′ is an organic group containing an alpha amino carboxylate, a    hydroxamate, or a catechol. Derivatized nanoparticles useful for    practice of the invention are described in detail in U.S. patent    application Ser. No. 10/822,940 filed Apr. 13, 2004 entitled    DERIVATIZED NANOPARTICLE COMPRISING METAL-ION SEQUESTRANT by    Joseph F. Bringley.

In a preferred embodiment the metal-ion sequestering agent isimmobilized in a polymeric layer, and the polymeric layer contacts thefluid contained therein. The metal-ion sequestrant may be formedintegrally within the materials comprising the bottle or may becontained within a polymeric layer directly in contact with the beverageor liquid nutrient. It is preferred that the polymer is permeable towater. It is preferred that the metal-ion sequestering agent comprisesare 0.1 to 50.0% by weight of the polymer. Polymers useful for practiceof the invention are described in detail in U.S. patent application Ser.No. 10/823,453 filed Apr. 13, 2004 entitled ARTICLE FOR INHIBITINGMICROBIAL GROWTH by Joseph F. Bringley et al.

In a preferred embodiment, the metal-ion sequestering agent comprises analpha amino carboxylate, a hydroxamate, or a catechol functional group.Metal-ion sequestrants suitable for practice of the invention includeethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetic aciddisodium salt, diethylenetriaminepentaacetic acid (DTPA),Hydroxylpropylenediaminetetraacetic acid (DPTA), nitrilotriacetic acid,triethylenetetraaminehexaacetic acid, N,N′-bis(o-hydroxybenzyl)ethylenediamine-N,N′diacteic acid, andethylenebis-N,N′-(2-o-hydroxyphenyl)glycine, acetohydroxamic acid, anddesferroxamine B (the iron chelating drug desferal), catechol,disulfocatechol, dimethyl-2,3-dihydroxybenzamide, mesitylenecatecholamide (MECAM) and derivatives thereof,1,8-dihydroxynaphthalene-3,6-sulfonic acid, and2,3-dihydroxynaphthalene-6-sulfonic acid, and siderophores moleculesnaturally synthesized by micro-organisms which have a very high affinityfor Fe. Metal-ion sequestering agents suitable for use in the inventionare described at length in U.S. patent application Ser. No. 10/822,940filed Apr. 13, 2004 entitled DERIVATIZED NANOPARTICLE COMPRISINGMETAL-ION SEQUESTRANT by Joseph F. Bringley et al.

The antimicrobial active material of antimicrobial agent may be selectedfrom a wide range of known antibiotics and antimicrobials. Anantimicrobial material may comprise an antimicrobial ion, moleculeand/or compound, metal ion exchange materials exchanged or loaded withantimicrobial ions, molecules and/or compounds, ion exchange polymersand/or ion exchange latexes, exchanged or loaded with antimicrobialions, molecules and/or compounds. Suitable materials are discussed in“Active Packaging of Food Applications” A. L. Brody, E. R. Strupinskyand L. R. Kline, Technomic Publishing Company, Inc. Pennsylvania (2001).Examples of antimicrobial agents suitable for practice of the inventioninclude benzoic acid, sorbic acid, nisin, thymol, allicin, peroxides,imazalil, triclosan, benomyl, metal-ion release agents, metal colloids,anhydrides, and organic quaternary ammonium salts. Preferredantimicrobial reagents are metal ion exchange reagents such as silversodium zirconium phosphate, silver zeolite, or silver ion exchange resinwhich are commercially available. The antimicrobial agent may beprovided in a layer 15 having a thickness “y” of between 0.1 microns and100 microns, preferably in the range of 1.0 and 25 microns.

In another preferred embodiment, the antimicrobial agent comprising acomposition of matter comprising an immobilized metal-ionsequestrant/antimicrobial comprising a metal-ion sequestrant that has ahigh stability constant for a target metal ion and that has attachedthereto an antimicrobial metal-ion, wherein the stability constant ofthe metal-ion sequestrant for the antimicrobial metal-ion is less thanthe stability constant of the metal-ion sequestrant for the targetmetal-ion. These are explained in detail in U.S. patent application Ser.No. 10/868,626 filed Jun. 15, 2004 entitled AN IRON SEQUESTERINGANTIMICROBIAL COMPOSITION by Joseph F. Bringley et al.

In a preferred embodiment, the antimicrobial agent comprising a metalion exchange material, is exchanged with at least one antimicrobialmetal ion selected from silver, copper, gold, nickel, tin or zinc.

Referring to FIG. 3, there is illustrated an embodiment of a fluidcontainer 5 made in accordance with the present invention. The container5, which in the embodiment illustrated is a bottle, is made of amaterial that comprises a barrier layer 22, an outer polymeric layer 20and an inner polymeric layer 40 between said barrier layer 22 and outerpolymeric layer 20. The inner polymeric layer 22 contains a metal-ionsequestrant 35. The barrier layer 22 preferably does not contain themetal-ion sequestrant 35. The outer layer 20 may provide severalfunctions including improving the physical strength and toughness of thearticle and resistance to scratching, marring, cracking, etc. However,the primary purpose of the barrier layer 22 is to provide a barrierthrough which micro-organisms 25 present in the contained fluid cannotpass. It is important to limit or eliminate, in certain applications,the direct contact of micro-organisms 25 with the metal-ion sequestrant35 or the layer containing the metal-ion sequestrant 35, since manymicro-organisms 25, under conditions of iron deficiency, maybio-synthesize molecules which are strong chelators for iron and othermetals. These bio-synthetic molecules are called “siderophores” andtheir primary purpose is to procure iron for the micro-organisms 25.Thus, if the micro-organisms 25 are allowed to directly contact themetal-ion sequestrant 35, they may find a rich source of iron there andbegin to colonize directly at these surfaces. The siderophores producedby the micro-organisms may compete with the metal-ion sequestrant forthe iron (or other bio-essential metal) at their surfaces. However theenergy required for the organisms to adapt their metabolism tosynthesize these siderophores will impact significantly their growthrate. Thus, one object of the invention is to lower growth rate oforganisms in the contained liquid. Since the barrier layer 22 of theinvention does not contain the metal-ion sequestrant 35, and becausemicro-organisms are large, the micro-organisms may not pass or diffusethrough the barrier layer 22. The barrier layer 22 thus prevents contactof the micro-organisms with the polymeric layer 40 containing themetal-ion sequestrant 35 of the invention. It is preferred that thebarrier layer 22 is permeable to water. It is preferred that the barrierlayer 22 has a thickness “x” in the range of 0.1 microns to 10.0microns. It is preferred that microbes are unable to penetrate, todiffuse or pass through the barrier layer 22. Sequestrant 35 with asequestered metal ion is indicated by numeral 35′.

Still referring again to FIG. 3, the enlarged sectioned view of thefluid container 5 shown in 3, illustrates a bottle having barrier layer22, which is in direct contact with the liquid nutrient 10, an innerpolymeric layer 40 and an outer polymeric layer 20. However, the bottleof FIG. 2 comprises an inner polymeric layer 15 that does not containany metal-ion sequestering agents. In the prior art bottle illustratedin FIG. 2, the micro-organisms 25 are free to gather the “free” ironions 30. In the example shown in FIG. 3, the inner polymer 40 containsan immobilized metal-ion sequestering agent 35 such as EDTA. In orderfor the metal-ion sequestering agent 35 to work properly, the innerpolymer 40 containing the metal-ion sequestering agent 35 must bepermeable to aqueous media. Preferred polymers for layers 22 and 40 ofthe invention are polyvinyl alcohol, cellophane, water-basedpolyurethanes, polyester, nylon, high nitrile resins,polyethylene-polyvinyl alcohol copolymer, polystyrene, ethyl cellulose,cellulose acetate, cellulose nitrate, aqueous latexes, polyacrylic acid,polystyrene sulfonate, polyamide, polymethacrylate, polyethyleneterephthalate, polystyrene, polyethylene, polypropylene orpolyacrylonitrile. A water permeable polymer permits water to movefreely through the polymer 40 allowing the “free” iron ion 30 to reachand be captured by the agent 35. An additional barrier 22 may be used toprevent the micro-organism 25 from reaching the inner polymer material40 containing the metal-ion sequestering agent 35. Like the innerpolymer material 40, the barrier layer 22 must be made of awaterpermeable polymer as previously described. The micro-organism 25 istoo large to pass through the barrier 22 or the polymer 40 so it cannotreach the sequestered iron ion 30 now held by the metal-ion sequesteringagent 35. By using the metal-ion sequestering agents 35 to significantlyreduce the amount of “free” iron ions 30 in the liquid nutrient 10, thegrowth of the micro-organism 25 is eliminated or severely reduced.

In the embodiment shown in FIGS. 4, 5, and 6, the metal-ion sequesteringagent 35 is contained in the bottle cap 50 instead of on the insidesurface of the bottle 5. An inner portion 45 of the cap 50, which is inintimate contact with the liquid nutrient 10, is made of a hydrophilicpolymer 55 containing the metal-ion sequestering agent 35 such as EDTAas described above. In some situations, the bottle may need to be placedin the inverted position in order for the sequestrant to become incontact with the contained nutrient. The cap 50 may also have thebarrier layer 22 to further prevent the micro-organisms 25 from reachingthe sequestered “free” iron 30. In another embodiment (not shown) thecap sealing material could be an open cell foamed structure whose cellwalls are coated with the sequestering material.

In still another embodiment, the sequestering agent 35 may be in ahydrophilic polymeric insert 52 that is placed in the bottle 5 asillustrated in FIG. 4. The insert 52 may be instead of or in addition tothe sequestrant in the cap 50 or interior of the bottle. The insert 52is placed in the bottle 5 but unfolds making it too large to exit thebottle 5. In another version, the insert 52 is molded into the bottom ofthe bottle 5.

Referring to FIGS. 7 and 8, there is illustrated another modifiedembodiment of a container made in accordance with the present invention,like parts indicating like parts and operation as previously described.In this embodiment the metal-ion sequestering agent 35 is contained in aprojecting member 60 that extends from cap 50 into the bottle 5 so thatit will be in intimate contact with the liquid nutrient 10. In theembodiment, the projecting member is in the configuration of a strawthat can later be used to drink the liquid content in the bottle. Likethe hydrophilic polymer material lining of the inside of the bottle 5and bottle cap 50, the extension 60 or straw is made of a hydrophilicpolymer 65 containing the metal-ion sequestering agents 35 such as EDTAas described in FIG. 3. When the bottle 5 is filled with the liquidnutrient 10 such as an isotonic, and is capped, the straw 60 protrudesfrom the cap 50 into the solution 10 allowing the “free” iron ions 35 tobe sequestered from the liquid nutrient 10. The straw 60 may also havethe barrier layer 22 to further prevent the micro-organisms 25 fromreaching the sequestered “free” iron ions 30. The outer layer 20 mayalso be made of a material similar to barrier layer 22 so that “free”iron ions 30 can reach the sequestrant 35 from the outside of the straw60.

In the example shown the extension is a straw but the extension can beof any shape just as long as it extends into the food or beverageestablishing intimate contact.

Referring to FIGS. 9 and 10, there is illustrated another embodiment ofa bottle 5 made in accordance with the present invention. In thisembodiment, the metal-ion sequestering agent 35 is applied to the insidesurface 80 of the bottle 5 by spraying a metal-ion sequestering agent35, for example EDTA, on to the inside surface of the bottle through asupply tube 85 using a spherical shaped nozzle assembly 90. The nozzleassembly 90 is moved up and down in the direction of the arrow 95 whilethe metal-ion sequestering agent 35 is sprayed as indicated by thearrows 100. The method of applying coatings to glass, metal or plasticcontainers is well known to those skilled in the art. FIG. 10illustrates an enlarged partial cross sectional view of the portion ofthe bottle of FIG. 9 where the spray coating 105 of the ion sequesteringagent 35 has been applied. As previously discussed in FIG. 3, likenumerals indicate like parts and operations. It is of course understoodthat the inner layer containing the sequestrant may be applied or formedon the inside surface of the container in any appropriate manner. Thebottle 5 in this embodiment may be made of any appropriate plastic orglass material.

Referring to FIGS. 11 and 12, there is illustrated yet another modifiedcontainer 110 made in accordance with the present invention. Inparticular the container comprises juice/drink box 110 for containing aliquid beverage. The box 110 is made of a sheet material that comprisesinner layer 115, a middle layer 120 made of a hydrophobic polymermaterial, and an outer layer 125. The inner layer 115 is in directcontact with the liquid nutrient 10 and is made of a hydrophilic polymercontaining the metal-ion sequestering agent 35 such as EDTA as describedabove in FIG. 3. As previously discussed in FIG. 3, like numeralsindicate like parts and operations. The outer layer 125 may comprise afoil wrap.

Referring to FIGS. 13 and 14, there is illustrated yet another modifiedembodiment of a container 130 made in accordance with the presentinvention. In the embodiment, the container comprises a stand up pouch130. The pouch 130 comprises an inner layer 135 made of a hydrophilicpolymer material, and an outer layer 140. The outer layer 140 may bemade of a polymer such as Mylar™ with a metalized coating 145. The innerlayer 135 is in direct contact with the liquid nutrient 10 and is madeof a hydrophilic polymer containing the metal-ion sequestering agent 35such as EDTA as described above in FIG. 3. The stand up pouch 130 mayalso have the barrier layer 22 not shown to further prevent themicro-organisms 25 from reaching the sequestered “free” iron 30. Aspreviously discussed in FIG. 3, like numerals indicate like parts andoperations.

Referring to FIGS. 15 and 16, there is illustrated still anothermodified container made in accordance with the present invention. Inthis embodiment the container comprises a bag 150. The bag 150, which isintended to hold an aqueous material, comprises an inner layer 155 madeof a hydrophobic polymer material and an outer layer 160. The outerlayer 140 may be made of a polymer such as polyethylene terephthalate.The inner layer 155 is in direct contact with the aqueous material 155and is made of a hydrophilic polymer containing the metal-ionsequestering agent 35 such as EDTA as described above in FIG. 3. The bag150 may also have the barrier layer 22 not shown to further prevent themicro-organisms 25 from reaching the sequestered “free” iron 30. Aspreviously discussed in FIG. 3, like numerals indicate like parts andoperations.

The juice box 110, the pouch 130 and the bag 150 may be constructed froma base web 170 as illustrated in FIG. 17. After the base web 170 isformed, the hydrophilic layer 175 is applied via a coating assembly 180comprised of a reservoir 185, an applicator 190 and a drive mechanism(not shown) to form the hydrophilic inner layer 175 containing themetal-ion sequestering agent 35 as described above in FIG. 3. Othermethods of forming and of making webs and applying a coating such ascoextrusion maybe used. It is of course understood that any suitabletechnique or process may be used for applying a coating on supportingweb as long as the coating has the appropriate sequestrant.

Referring to FIGS. 18 and 19 there is illustrated a modified container220 made in accordance with the present invention. In this embodiment,the container 220 comprises a can. The can 200 is made of a metalmaterial such as aluminum or steel, and has a top and a bottom, whichmay or may not be made as separate piece. The can 200 may also have alining 205, which is in direct contact with the aqueous material 155 andintended to prevent corrosion of the metal by the contents of the can.The construction of metal cans is well known by one skilled in the art.The lining 205 may include a hydrophilic polymer containing themetal-ion sequestering agent 35 or have a hydrophilic polymer strip 210containing metal-ion sequestering agent 35 made as part of lining 205 ofthe can 200. The strip 210 may have a width “w” of between 1 millimeterand 30 millimeters and be spaced at intervals around the insidecircumference of the can 200 and a depth “d” of −1.0 to 10 micrometers.In still another embodiment, the sequestering agent 35 may be in ahydrophilic polymeric insert 52. The insert 52 is placed in the can 200but unfolds making it too large to exit the can 200. The insert 52 maybe simply placed on the bottom of the container or if desired secured tothe interior surface of the container in some fashion. The metal-ionsequestering agent performs as previously described above in FIG. 3.

Referring to FIG. 20, there is illustrated a cross-sectional view of afilter assembly 220 comprising an inlet port 225, an outlet port 230,and a filter 235. The filter 235 contains an immobilized metal-ionsequestering agent as previously described. As the solution flowsthrough the filter assembly 220 in the direction indicated by the arrows240, and through the filter 235, the metal ions in the solution aresequestered and removed by the metal-ion sequestering agent 245.

In a preferred embodiment using a filter assembly the solution would becontinuously circulated through the filter assembly for a period rangingfrom 30 minutes to 24 hours until the metal ion co-concentration isreduced to a level to minimize or eliminate microbial growth asdescribed in U.S. patent application Ser. No. 10/822,940 filed Apr. 13,2004 entitled DERIVATIZED NANOPARTICLE COMPRISING METAL-ION SEQUESTRANTby Joseph F. Bringley et al.

An example of removal is shown in the graph illustrated in FIG. 25.

Referring to FIG. 21, there is illustrated a cross sectional view of afluid bed ion exchange assembly 250 comprising a holding tank 255, aninlet port 260, an outlet port 265, and a fluid bed 270 containing ametal-ion sequestering material 275 made in accordance with the presentinvention. The solution 280 flows into the fluid bed ion exchangeassembly 250 via inlet port 260 as indicated by arrow 285 through themetal-ion sequestering material 275 in fluid bed 270 as indicated byarrows 290 and out the outlet port as indicated by arrow 295.

FIG. 22 is an enlarged partial view of a portion of the fluid bed 270containing a metal-ion sequestering material 275. An example of themetal-ion sequestering material 275 comprises a core material 300 and ashell material 305 made of the metal-ion sequestering agent 35 asdescribed in U.S. patent application Ser. No. 10/822,940 filed Apr. 13,2004 entitled DERIVATIZED NANOPARTICLE COMPRISING METAL-ION SEQUESTRANTby Joseph F. Bringley et al. As previously described above in FIG. 21,the solution 280 containing “free” metal ions 310 flows through thefluid bed 270 as indicated by the arrows 315. As the solution 280 flowsthrough the fluid bed 270 the shell material 305 made of the metal-ionsequestering agent 35 gathers the metal ions 320 removing them from thesolution, which then flow out through the outlet port 265.

In a preferred embodiment the flow rate through the bed will be suchthat the solution is in contact with the bed for 1 to 30 minutes duringwhich the metal ion concentration will be reduced by at least 80% andmicrobial growth will be minimized and/or eliminated.

For example removal by particles in a simulated fluid bed assembly isshown in the graph illustrated in FIG. 26.

While in the embodiments discussed, the iron sequestering agent orantimicrobial agent may be provided only on a portion of the contactingsurface of the bottle or other container. For example, but not limitedto, the agents may be provided only on the body portion of a bottle andnot the neck portion.

While in many of the embodiments illustrated a barrier layer is notdiscussed, it is to be understood that a barrier layer 22 may beprovided in any of the embodiments for preventing the microbes(micro-organism) from contacting the sequestrant.

EXAMPLES AND COMPARISON EXAMPLES

Materials:

Colloidal dispersions of silica particles were obtained from ONDEO NalcoChemical Company. NALCO® 1130 had a median particle size of 8 nm, a pHof 10.0, a specific gravity of 1.21 g/ml, a surface area of about 375m²/g, and a solids content of 30 weight percent.N-(trimethoxysilylpropyl ethylenediamine triacetic acid, trisodium saltwas purchased from Gelest Inc., 45% by weight in water.

Preparation of derivatized nanoparticles. To 600.00 g of silica NALCO®1130 (30% solids) was added 400.00 g of distilled water and the contentsmixed thoroughly using a mechanical mixer. To this suspension, was added49.4 g of N-(trimethoxysilyl) propylethylenediamine triacetic acid,trisodium salt in 49.4 g distilled water with constant stirring at arate of 5.00 ml/min. At the end of the addition the pH was adjusted to7.1 with the slow addition of 13.8 g of concentrated nitric acid, andthe contents stirred for an hour at room temperature. Particle sizeanalysis indicated an average particle size of 15 nm. The percent solidsof the final dispersion was 18.0%.

Preparation of the immobilized metal-ion sequestrant/antimicrobial:200.0 g of the above derivatized nanoparticles were washed withdistilled water via dialysis using a 6,000-8,000 molecular weight cutofffilter. The final ionic strength of the solution was less than 0.1millisemens. To the washed suspension was then added with stirring 4.54ml of 1.5 M AgNO₃ solution, to form the immobilized metal-ionsequestrant/antimicrobial.

Preparation of Polymeric Layers of Immobilized Metal-Ion Sequestrantsand Sequestrant/antimicrobials.

Coating 1 (comparison). A coating solution was prepared as follows: 8.8g of a 40% solution of the polyurethane Permax 220 (Noveon Chemicals)was combined with to 90.2 grams of pure distilled water and 1.0 g of a10% solution of the surfactant OLIN 10G was added as a coating aid. Themixture was then stirred and blade-coated onto a polymeric support usinga 150 micron doctor blade. The coating was then dried at 40-50° C., toproduce a film having 5.4 g/m² of polyurethane.

Coating 2. A coating solution was prepared as follows: 171.2 grams ofthe derivatized nanoparticles prepared as described above were combinedwith 64.8 grams of pure distilled water and 62.5 g of a 40% solution ofthe polyurethane Permax 220 (Noveon Chemicals). 1.5 g of a 10% solutionof the surfactant OLIN 10G was added as a coating aid. The mixture wasthen stirred and blade-coated onto a polymeric support using a 150micron doctor blade. The coating was then dried at 40-50° C., to producea film having 5.4 g/m² of the derivatized nanoparticles and 5.4 g/m² ofpolyurethane.

Coating 3. A coating solution was prepared as follows: 171.2 grams ofthe derivatized nanoparticles prepared as described above were combinedwith 33.5 grams of pure distilled water and 93.8 g of a 40% solution ofthe polyurethane Permax 220 (Noveon Chemicals). 1.5 g of a 10% solutionof the surfactant OLIN 10G was added as a coating aid. The mixture wasthen stirred and blade-coated onto a polymeric support using a 150micron doctor blade. The coating was then dried at 40-50° C., to producea film having 5.4 g/m² of the derivatized nanoparticles and 8.1 g/m² ofpolyurethane.

Coating 4. A coating solution was prepared as follows: 138.9 grams ofthe derivatized nanoparticles prepared as described above were combinedwith 97.1 grams of pure distilled water and 62.5 g of a 40% solution ofthe polyurethane Permax 220 (Noveon Chemicals). 1.5 g of a 10% solutionof the surfactant OLIN 10G was added as a coating aid. The mixture wasthen stirred and blade-coated onto a polymeric support using a 150micron doctor blade. The coating was then dried at 40-50° C., to producea film having 4.4 g/m² of the derivatized nanoparticles and 5.4 g/m² ofpolyurethane.

Coating 5. A coating solution was prepared as follows: 12.8 grams of theimmobilized metal-ion sequestrant/antimicrobial suspension prepared asdescribed above was combined with to 77.4 grams of pure distilled waterand 8.8 g of a 40% solution of the polyurethane Permax 220 (NoveonChemicals). 1.0 g of a 10% solution of the surfactant OLIN 10G was addedas a coating aid. The mixture was then stirred and blade-coated onto apolymeric support using a 150 micron doctor blade. The coating was thendried at 40-50° C., to produce a film having 2.7 g/m² of the immobilizedmetal-ion sequestrant/antimicrobial, 0.06 g/m² silver-ion and 5.4 g/m ofpolyurethane.

Now referring to FIGS. 23 and 24, there is illustrated anotherembodiment of the web 170 shown in FIG. 17 used for the manufacture ofthe box 110, pouch 130 or bag 150 made in accordance with the presentinvention. In the embodiment shown an electro-photographic deviceemploys magnetic brush technology to apply toner particles 400 comprisedof the metal-ion sequestering agents 35 in a predetermined pattern 475to the web 170 forming a metal-ion sequestering web 410. Thepredetermined pattern may comprise the entire surface of said web 170 orselected locations on said web 170. In the example shown theelectro-photographic device uses the web 170, which is driven in thedirection indicated by the arrow 405 by drive roller assembly 490. Theweb 170 is charged with a high voltage corona via a charger 420 andselectively discharged with light from a light source 430 such as an LEDarray to form an electrostatic pattern 440. The electrostatic pattern isdeveloped with the negatively charged toner particles by a magneticbrush 460 composed of the negatively charged toner particles andmagnetic carrier particles (not shown) on a magnetic brush 460. Thecharged toner particles 475 are then fused to the web 170 by heatedrollers 490 to form a fused pattern 480 as the web 170 moves in thedirection of the arrow 405. By laying down a pattern the edges 500 shownin FIG. 23 may be left clean to facilitate sealing of the box 110, pouch130 or bag 150. As in the case of an electro-photographic printer theamount or density of the toner particles transferred to the web can becontrolled by the amount of charge placed on the web. Using thistechnique, which is understood by those skilled in the art, the amountor density of metal-ion sequestering agents 35 in the form of tonerparticles can likewise be controlled. This allows for providing themetal-ion sequestering agent at a designated location and at a desireddensity on web 170. Thus, the web may be designed to meet the variousneeds of the user. For example, if more that one designated metal-ion isto be treated, more than one type metal-ion sequestering agent can beapplied to web. In addition the density may be varied to adjust to theamount to which the designated metal ion is present in the liquidnutrient allowing for efficient use of both metal ion sequesteringagents. In addition to providing different type metal-ion sequesteringagents other type agents may be applied in combination, for example, theelectro-photographic device may employ magnetic brush technology toapply toner particles 400 comprised of a metal-ion sequestering agentand an antimicrobial agent, as previously described, in a pattern 475 onto the web 170. The web 170 may be made of any suitable material and ofany suitable construction. For example but not by way of limitation, web170 may be made of nylon, polyester, natural or synthetic fabricmaterial, plastic, and may be continuous, or of a fabric type weave ofone or more material fibers. It is important that the material allow forthe adhering of the metal-ion sequestering agent and allow appropriateinteraction with the liquid agent.

TESTING METHODOLOGY

A test similar to ASTM E 2108-01 was conducted where a piece of acoating of known surface area was contacted with a solution inoculatedwith micro-organisms. In particular a piece of coating 1×1 cm was dippedin 2 ml of growth medium (Trypcase Soy Agar 1/10), inoculated with2000CFU of Candida albicans (ATCC-1023) per ml. Special attention wasmade to all reagents to avoid iron contamination with the final solutionhaving an iron concentration of 80 ppb before contact with the coating.

Micro-organism numbers in the solution were measured daily by thestandard heterotrophic plate count method.

The bar graph shown in FIG. 27 demonstrates the effectiveness of theinventive examples. The yeast population which was exposed to thecomparison coating 1 (which contained no derivatized nanoparticles)showed a growth factor of one thousand during 48 hours (a 1000-foldincrease in population). The yeast population which was exposed to theexample coatings 2-4 (containing derivatized nanoparticles) showedgrowth factors of only 1-4. This is indicative of a fungostatic orbio-static effect in which the population of organisms is kept at aconstant or near constant level, even in the presence of a mediumcontaining adequate nutrient level. The yeast population which wasexposed to the example coating 5 (derivatized nanoparticles that hadbeen ion exchanged with silver ion—a known antimicrobial) showed afungicidal effect (the yeast were completely eliminated). The low levelof silver when coated by itself without the nanoparticles would not beexpected to exhibit this complete fungicidal effect, and there appearsto be a synergistic effect between the iron sequestration and therelease of antimicrobial silver.

As can be seen from the bar graph illustrated in FIG. 27, significantimproved results may be obtained when a metal-ion sequestering agent isused in conjunction with an antimicrobial agent. The combined agentsreduced the level of microbes to lower level than when first introducedand then maintained the reduced level of microbes in the liquidnutrient.

The invention has been described in detail with particular reference tocertain preferred embodiments thereof but it will be understood thatvariations and modifications can be effected within the spirit and scopeof the invention.

PARTS LIST

-   5 fluid container/bottle-   10 liquid nutrient-   15 inner polymeric layer-   20 outer polymeric layer-   22 barrier layer-   25 micro-organism-   30 “free” iron ion-   35 metal-ion sequestering agents-   35′ metal-ion sequestering agent with a sequestered metal ion-   40 hydrophilic polymer-   45 inner portion-   50 bottle cap-   52 insert-   55 hydrophilic polymer-   60 extension (straw)-   65 hydrophilic polymer-   80 inside surface-   85 supply tube-   90 spherical shaped nozzle assembly-   95 arrow-   100 arrow-   105 spray coating-   110 juice box-   115 inner layer-   120 middle layer-   125 outer layer-   130 pouch-   135 inner layer-   140 outer layer-   145 coating-   150 bag-   155 aqueous material-   160 inner layer-   165 outer layer-   170 base web-   175 hydrophilic layer-   180 coating assembly-   185 reservoir-   190 applicator-   200 can-   205 lining-   210 strip-   220 filter assembly-   225 inlet port-   230 outlet port-   235 filter-   240 arrow-   250 fluid bed ion exchange assembly-   255 holding tank-   260 inlet port-   265 outlet port-   270 fluid bed-   275 sequestering material-   280 solution-   285 arrow-   290 arrow-   295 arrow-   300 core material-   305 shell material-   310 “free” metal ions-   315 arrows-   320 gathered metal ions

1. A method for inhibiting growth of microbes in a liquid having a pHequal to or greater than about 2.5, comprising the steps of: a.providing a filter assembly for filtering said liquid, said filterassembly having a filter having a metal-ion sequestering agent forremoving designated metal ions from said liquid, said filter assemblyhaving an inlet and an outlet for allowing said liquid to enter andleave said filter assembly, wherein the filter comprises a barrier layerand a polymeric layer, the polymeric layer contains the sequesteringagent and is disposed between a surface of the filter and the barrierlayer, and wherein the barrier layer is permeable to water so that thewater contacts the sequestering agent but microbes cannot pass ordiffuse through the barrier layer; and b. causing said liquid to entersaid filter assembly through said inlet pass through said filter and outsaid outlet for a sufficient time so as to substantially reduce theamount of the designated metal ion from said liquid, wherein the growthof said microbes is inhibited.
 2. The method of claim 1 wherein saidsufficient time comprises from about 1 to 30 minutes.
 3. The method ofclaim 1 wherein a concentration of said designated metal ions is reducedby at least 80%.
 4. The method of claim 1 wherein said metal-ionsequestering agent has a stability constant greater than 10¹⁰ with iron(III).
 5. The method of claim 1 wherein said sequestering agent has ahigh-affinity for biologically important metal ions including Mn, Mg,Zn, Cu and Fe.
 6. The method of claim 1 wherein said sequestering agenthas a high-selectivity for biologically important metal ions includingMn, Mg, Zn, Cu and Fe.
 7. The method of claim 1 wherein saidsequestering agent has a high-selectively for certain metal ions but alow-affinity for at least one other ion.
 8. The method of claim 7wherein said certain metal ions comprises Mn, Mg, Zn, Cu and Fe and saidat least one other ion comprises calcium.
 9. The method of claim 1wherein said metal-ion sequestering agent has a stability constantgreater than 10²⁰ with iron (III).
 10. The method of claim 1 whereinsaid metal-ion sequestering agent has a stability constant greater than10³⁰ with iron (III).
 11. The method of claim 1 wherein said metal-ionsequestering agent comprises derivatized nanoparticles comprisinginorganic nanoparticles having an attached metal-ion sequestrant,wherein said inorganic nanoparticles have an average particle size ofless than 200 nm and the derivatized nanoparticles have a stabilityconstant greater than 10¹⁰ with iron (III).
 12. The method of claim 11wherein said inorganic nanoparticles have an average particle size ofless than 100 nm.
 13. The method of claim 11 wherein said metal-ionsequestrant is attached to the nanoparticle by reacting the nanoparticlewith a silicon alkoxide intermediate of the sequestrant having thegeneral formula:Si(OR)_(4-x)R′_(x); wherein x is an integer from 1 to 3; R is an alkylgroup; and R′ is an organic group containing an alpha amino carboxylate,a hydroxamate, or a catechol.
 14. The method of claim 1 wherein saidmetal-ion sequestering agent is immobilized in the polymeric layer, andthe polymeric layer contacts the liquid contained therein.
 15. Themethod of claim 14 wherein the polymeric layer is permeable to water.16. The method of claim 14 wherein the metal-ion sequestering agentcomprises about 0.1 to about 50.0% by weight of the polymeric layer. 17.The method of claim 1 wherein said metal-ion sequestering agentcomprises an alpha amino carboxylate, a hydroxamate, or a catecholfunctional group.
 18. The method of claim 1 wherein said metal-ionsequestering agent comprises a naturally synthesized siderophoremolecule.
 19. The method of claim 1 wherein the barrier layer has athickness in the range of about 0.1 microns to about 10.0 microns. 20.The method of claim 1 where said liquid comprises a beverage.
 21. Themethod of claim 1 wherein said filter further includes an antimicrobialagent for reducing and/or maintaining an amount of microbes in saidliquid to a prescribed condition.
 22. A method for inhibiting growth ofmicrobes in a liquid having a pH equal to or greater than about 2.5,comprising the steps of: a. providing a filter assembly for filteringsaid liquid, said filter assembly having a filter having a metal-ionsequestering agent for removing designated metal ions from said liquid,said filter assembly having an inlet and an outlet for allowing saidliquid to enter and leave said filter assembly; and b. causing saidliquid to enter said filter assembly through said inlet pass throughsaid filter and out said outlet for a sufficient time so as tosubstantially reduce the amount of the designated metal ion from saidliquid, wherein the growth of microbes is inhibited, said metal-ionsequestering agent comprises derivatized nanoparticles comprisinginorganic nanoparticles having an attached metal-ion sequestrant, saidinorganic nanoparticles have an average particle size of less than 200nm, and wherein the derivatized nanoparticles have a stability constantgreater than 10¹⁰ with iron (III).
 23. The method of claim 22 whereinsaid sufficient time comprises from about 1 to 30 minutes.
 24. Themethod of claim 22 wherein a concentration of said designated metal ionsis reduced by at least 80%.
 25. The method of claim 22 wherein saidmetal-ion sequestering agent is immobilized on a surface of said filterand has a stability constant greater than 10¹⁰ with iron (III).
 26. Themethod of claim 22 wherein said sequestering agent is immobilized on asurface of said filter and has a high-affinity for biologicallyimportant metal ions including Mn, Mg, Zn, Cu and Fe.
 27. The method ofclaim 22 wherein said sequestering agent is immobilized on a surface ofsaid filter and has a high-selectivity for biologically important metalions including Mn, Mg, Zn, Cu and Fe.
 28. The method of claim 22 whereinsaid sequestering agent has a high-selectively for certain metal ionsbut a low-affinity for at least one other ion.
 29. The method of claim28 wherein said certain metal ions comprises Mn, Mg, Zn, Cu and Fe andsaid at least one other ion comprises calcium.
 30. The method of claim22 wherein said metal-ion sequestering agent is immobilized on a surfaceof said filter and has a stability constant greater than 10²⁰ with iron(III).
 31. The method of claim 22 wherein said metal-ion sequesteringagent is immobilized on a surface of said filter and has a stabilityconstant greater than 10³⁰ with iron (III).
 32. The method of claim 22wherein said metal-ion sequestering agent is immobilized in a polymericlayer, and the polymeric layer contacts the liquid contained therein.33. The method of claim 32 wherein the polymeric layer is permeable towater.
 34. The method of claim 32 wherein the metal-ion sequesteringagent comprises about 0.1 to about 50.0% by weight of the polymericlayer.
 35. The method of claim 22 wherein said inorganic nanoparticleshave an average particle size of less than 100 nm.
 36. The method ofclaim 22 wherein said metal-ion sequestering agent comprises an alphaamino carboxylate, a hydroxamate, or a catechol functional group. 37.The method of claim 22 wherein said metal-ion sequestering agentcomprises a naturally synthesized siderophore molecule.
 38. The methodof claim 22 wherein said metal-ion sequestrant is attached to thenanoparticle by reacting the nanoparticle with a silicon alkoxideintermediate of the sequestrant having the general formula:Si(OR)_(4-x)R′_(x); wherein x is an integer from 1 to 3; R is an alkylgroup; and R′ is an organic group containing an alpha amino carboxylate,a hydroxamate, or a catechol.
 39. The method of claim 22 furthercomprising a barrier layer and a polymeric layer, wherein the polymericlayer is between a surface of the filter and the barrier layer andwherein the barrier layer does not contain the derivatizednanoparticles.
 40. The method of claim 39 wherein the barrier layer ispermeable to water.
 41. The method of claim 39 wherein the barrier layerhas a thickness in the range of about 0.1 microns to about 10.0 microns.42. The method of claim 39 wherein microbes cannot pass or diffusethrough the baffler layer.
 43. The method of claim 22 where said liquidcomprises a beverage.
 44. The method of claim 22 wherein said filterfurther includes an antimicrobial agent for reducing and/or maintainingan amount of microbes in said liquid to a prescribed condition.